Immuno-molecules containing viral proteins, compositions thereof and methods of using

ABSTRACT

An immuno-molecule which comprises a soluble human MHC class I effector domain; and an antibody targeting domain which is linked to the soluble human MHC class I effector domain, methods of making same and uses thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/482,532 filed on Sep. 17, 2004, which is a National Phase of PCTPatent Application No. PCT/IL02/00478 filed on Jun. 18, 2002, which is acontinuation of U.S. patent application Ser. No. 10/108,511 filed onMar. 29, 2002, now abandoned, which claims the benefit of priority ofU.S. Provisional Patent Application No. 60/298,915 filed on Jun. 19,2001. The contents of the above applications are all incorporated hereinby reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a novel concept in immunotherapy, bywhich deception of the immune system results in specific and mostefficient destruction of cells of interest, cancer cells in particular.

There is strong evidence that tumor progression in cancer patients iscontrolled by the immune system. This conclusion is based onobservations that tumor progression is often associated with secretionof immune suppressive factors and/or downregulation of MHC class Iantigen presentation functions (1-5). The inference is that tumors musthave elaborated strategies to circumvent an apparently effective immuneresponse. Importantly, a tumor-specific immune response can be detectedin individuals (6-8).

The apparent inefficiency of anti tumor immune responses that results infailure to combat the disease laid the foundation to current concepts ofimmunotherapy. It is suggested that boosting the anti-tumor immuneresponse by deliberate vaccination or by other immunotherapeuticapproaches may increase the potential benefits of immune-based therapies(6, 9-11).

The MHC class I-restricted CD8 cytotoxic T cell (CTL) effector arm ofthe adaptive immune response is best equipped to recognize the tumor asforeign and initiate the cascade of events resulting in tumordestruction (12,13). Therefore, the most attractive approach in cancerimmunotherapy is centered on vaccination strategies designed to enhancethe CTL arm of the antitumor response and consequently overcome themechanisms of tumor escape from CTL (9-11).

One of the best-studied escape mechanisms by which tumor cells evadeimmune attack is by downregulation of the MHC class I molecules whichare the antigens recognized by CTLs (1-5,14).

Mutations along the class I presentation pathway should be the simplestway for tumors to escape CTL-mediated elimination since it can beachieved by one or two mutational events (two mutations to inactivateboth alleles or one mutation to create a dominant negative inhibitor)(1-3).

Downregulation of MHC class I expression is frequently observed in humantumors, and is particularly pronounced in metastatic lesions (3, 14-17).This is circumstantial but nevertheless compelling evidence of the roleof CTL in controlling tumor progression in cancer patients. MHC class Iexpression has been mainly analyzed in surgically removed tumorspecimens using immunohistochemical methods (14-15). Partial reductionor complete loss of MHC have been reported, encompassing all MHCmolecules or limited to particular alleles (14-15). MHC loss can be seenin some but not all lesions of the same patient. Downregulation of MHCclass I expression has been attributed to mutations in β2-microglobulin((β2-m), transporter associated with antigen presentation (TAP)proteins, or the proteosomal LMP-2 and LMP-7 proteins (2, 18-21).Additional evidence implicating loss of MHC class I expression as amechanism for tumor escape from CTL-mediated elimination comes from alongitudinal study of a melanoma patient. Tumor cells removed duringinitial surgery presented nine different antigens restricted to fourseparate HLA class I alleles to CTL clones established from the patient(1). The patient remained disease free for 5 years after which ametastasis was detected. Notably, a cell line established from themetastatic lesion had lost all four alleles that had previously beenshown to present melanoma antigens.

Thus, the downregulation of class I MHC molecule is a severe limitingproblem for cancer immunotherapy and the application of anti-cancervaccines. There is thus a widely recognized need for, and it would behighly advantageous to have, an novel approach of immunotherapy devoidof the above limitations, namely an approach of immunotherapy which isindependent of the level of expression of MHC class I molecules bycancer cells.

SUMMARY OF THE INVENTION

The MHC class I-restricted CD8 cytotoxic T cell (CTL) effector arm ofthe adaptive immune response is best equipped to recognize tumor cellsas foreign and initiate the cascade of events resulting in tumordestruction. However, tumors have developed sophisticated strategies toescape immune effector mechanisms, of which the best-studied is bydownregulation of MHC class I molecules which are the antigensrecognized by CTLs.

To overcome this and develop new approaches for immunotherapy, and whilereducing the present invention to practice, a recombinant molecule wasconstructed in which a single-chain MHC is specifically targeted totumor cells through its fusion to cancer specific-recombinant antibodyfragments or a ligand that binds to receptors expressed by tumor cells.As an exemplary molecule of the present invention, a single-chain HLA-A2molecule was genetically fused to the variable domains of an anti IL-2receptor α subunit-specific humanized antibody, anti-Tac (aTac). Theconstruct, termed B2M-aTac(dsFv) was expressed in E. coli and functionalmolecules were produced by in vitro refolding in the presence ofHLA-A2-restricted antigenic peptides. Flow cytometry studies revealedthe ability to decorate antigen-positive, HLA-A2-negative human tumorcells with HLA-A2-peptide complexes in a manner that was entirelydependent upon the specificity of the targeting antibody fragment. Mostimportantly, B2M-aTac(dsFv)-mediated coating of target tumor cells madethem susceptible for efficient and specific HLA-A2-restricted, melanomagp100 peptide-specific CTL-mediated lysis. These results demonstrate theconcept that antibody-guided tumor antigen-specific targeting ofMHC-peptide complexes on tumor cells can render them susceptible andpotentiate CTL killing. This novel approach now opens the way for thedevelopment of new immounotherapeutic strategies based on antibodytargeting of natural cognate MHC ligands and CTL-based cytotoxicmechanisms.

Hence, while reducing the present invention to practice a novel strategywas developed to re-target class I MHC-peptide complexes on the surfaceof tumor cells in a way that is independent of the extent of class I MHCexpression by the target tumor cells. To this end, in one embodiment ofthe present invention, two arms of the immune system were employed infusion. One arm, the targeting moiety, comprises tumor-specificrecombinant fragments of antibodies directed to tumor or differentiationantigens which have been used for many years to target radioisotopes,toxins or drugs to cancer cells (22, 23). The second, effector arm, is asingle-chain MHC molecule (scMHC) composed of human β2-microglobulinlinked to the three extracellular domains of the HLA-A2 heavy chain (24,25, WO 01/72768). By connecting the two molecules into a singlerecombinant gene and expressing the gene. The new molecule is expressedefficiently in E. coli and produced, for example, by in vitro refoldingin the presence of HLA-A2-restricted peptides. This approach, as shownherein, renders the target tumor cells susceptible to lysis by cytotoxicT cells regardless of their MHC expression level and thus may beemployed as a new approach to potentiate CTL-mediated anti-tumorimmunity. This novel approach will lead to the development of a newclass of recombinant therapeutic agents capable of selective killing andelimination of tumor cells utilizing natural cognate MHC ligands andCTL-based cytotoxic mechanisms.

According to one aspect of the present invention there is provided animmuno-molecule comprising: a soluble human MHC class I effector domain;and a targeting domain being linked to the soluble human MHC class Ieffector domain.

Thus, according to another aspect of the present invention there isprovided a nucleic acid construct encoding an immuno-molecule, theconstruct comprising: a first polynucleotide encoding a soluble humanMHC class I effector domain; and a second polynucleotide encoding atargeting domain; the first polynucleotide and the second polynucleotideare selected and being joined such that the soluble human MHC class Ieffector domain and the antibody targeting domain are translationallyfused optionally via a peptide linker in-between.

According to still another aspect of the present invention there isprovided a nucleic acid construct encoding an immuno-molecule, theconstruct comprising: a first polynucleotide encoding a soluble humanMHC class I effector domain; and a second polynucleotide encoding avariable region of one of a light chain or a heavy chain of an antibodytargeting domain; the first polynucleotide and the second polynucleotideare selected and being joined such that the soluble human MHC class Ieffector domain and the variable region of the one of the light chainand heavy chain of the antibody targeting domain are translationallyfused optionally via a peptide linker in-between; and a thirdpolynucleotide encoding the other of the one of the light chain andheavy chain of the antibody targeting domain.

According to an additional aspect of the present invention there isprovided a nucleic acid construct system comprising: a first nucleicacid construct which comprises: a first polynucleotide encoding asoluble human MHC class I effector domain; and a second polynucleotideencoding a variable region of one of a light chain or a heavy chain ofan antibody targeting domain; the first polynucleotide and the secondpolynucleotide are selected and being joined such that the soluble humanMHC class I effector domain and the variable region of the one of thelight chain and heavy chain of the antibody targeting domain aretranslationally fused optionally via a peptide linker in-between; asecond nucleic acid construct which comprises: a third polynucleotideencoding the other of the one of the light chain and heavy chain of theantibody targeting domain.

According to a further aspect of the present invention there is provideda method of selectively killing a cell in a patient, the cell presentingan antigen (e.g., a receptor), the method comprising administering tothe patient an immuno-molecule which comprises: a soluble human MHCclass I effector domain complexed with an MHC-restricted peptide; and atargeting domain being linked to the soluble human MHC class I effectordomain, the targeting domain being for selectively binding to theantigen; whereby, the soluble human MHC class I effector domaincomplexed with the MHC-restricted peptide initiates a CTL mediatedimmune response against the cell, thereby selectively killing the cellin vivo.

According to further features in preferred embodiments of the inventiondescribed below, the targeting domain is an antibody targeting domain.

According to still further features in the described preferredembodiments the targeting domain is a ligand targeting domain.

According to still further features in the described preferredembodiments the ligand targeting domain is selected from the groupconsisting of PDGF, EGF, KGF, TGF, IL-2, IL-3, IL-4, IL-6, VEGF and itsderivatives, TNF.

According to still further features in the described preferredembodiments the soluble human MHC class I effector domain and theantibody targeting domain are translationally fused, optionally with atranslationally fused peptide linker in-between.

According to still further features in the described preferredembodiments the antibody targeting domain comprises a variable region ofa light chain of an antibody linked to the effector domain.

According to still further features in the described preferredembodiments the variable region of the light chain of the antibody andthe effector domain are translationally fused, optionally with atranslationally fused peptide linker in-between.

According to still further features in the described preferredembodiments the antibody targeting domain further comprises a variableregion of a heavy chain of an antibody linked to the variable region ofthe light chain of the antibody.

According to still further features in the described preferredembodiments the variable region of the heavy chain of the antibody andthe variable region of the light chain of the antibody aretranslationally fused, optionally with a translationally fused peptidelinker in-between.

According to still further features in the described preferredembodiments the variable region of the heavy chain of the antibody islinked to the variable region of the light chain of the antibody via apeptide linker.

According to still further features in the described preferredembodiments the variable region of the heavy chain of the antibody islinked to the variable region of the light chain of the antibody via atleast one S—S bond.

According to still further features in the described preferredembodiments the antibody targeting domain comprises a variable region ofa heavy chain of an antibody linked to the effector domain.

According to still further features in the described preferredembodiments the variable region of the heavy chain of the antibody andthe effector domain are translationally fused, optionally with atranslationally fused peptide linker in-between.

According to still further features in the described preferredembodiments the antibody targeting domain further comprises a variableregion of a light chain of an antibody linked to the variable region ofthe heavy chain of the antibody.

According to still further features in the described preferredembodiments the variable region of the light chain of the antibody andthe variable region of the heavy chain of the antibody aretranslationally fused, optionally with a translationally fused peptidelinker in-between.

According to still further features in the described preferredembodiments the variable region of the light chain of the antibody islinked to the variable region of the heavy chain of the antibody via apeptide linker.

According to still further features in the described preferredembodiments the variable region of the light chain of the antibody islinked to the variable region of the heavy chain of the antibody via atleast one S—S bond.

According to still further features in the described preferredembodiments the antibody targeting domain is capable of binding to atumor associated antigen.

According to still further features in the described preferredembodiments the antibody targeting domain is capable of binding to atumor specific antigen.

According to still further features in the described preferredembodiments the soluble human MHC class I effector domain comprises afunctional human β-2 microglobulin and a functional human MHC class Iheavy chain linked thereto.

According to still further features in the described preferredembodiments the functional human MHC class I heavy chain comprisesdomains α1-3.

According to still further features in the described preferredembodiments the functional human β-2 microglobulin and the functionalhuman MHC class I heavy chain are translationally fused, optionally witha translationally fused peptide linker in-between.

According to still further features in the described preferredembodiments the soluble human MHC class I effector domain furthercomprises a MHC-restricted peptide.

According to still further features in the described preferredembodiments the MHC-restricted peptide is linked to the functional humanβ-2 microglobulin.

According to still further features in the described preferredembodiments the MHC-restricted peptide and the functional human β-2microglobulin are translationally fused, optionally with atranslationally fused peptide linker in-between.

According to still further features in the described preferredembodiments the MHC-restricted peptide is complexed with the functionalhuman MHC class I heavy chain.

According to still further features in the described preferredembodiments the MHC-restricted peptide is derived from a commonpathogen.

According to still further features in the described preferredembodiments the MHC-restricted peptide is derived from a pathogen forwhich there is an active vaccination.

According to still further features in the described preferredembodiments the MHC-restricted peptide is derived from a tumorassociated or specific antigen.

According to further features in preferred embodiments of the inventiondescribed below, any of the nucleic acid constructs described herein,further comprising at least one cis acting regulatory sequence operablylinked to the coding polynucleotides therein.

According to still further features in the described preferredembodiments the cis acting regulatory sequence is functional inbacteria.

According to still further features in the described preferredembodiments the cis acting regulatory sequence is functional in yeast.

According to still further features in the described preferredembodiments the cis acting regulatory sequence is functional in animalcells.

According to still further features in the described preferredembodiments the cis acting regulatory sequence is functional in plantcells.

According to still another aspect of the present invention there isprovided a transformed cell comprising any of the nucleic acidconstructs or the nucleic acid construct system described herein.

According to further features in preferred embodiments of the inventiondescribed below, the cell is a eukaryotic cell selected from the groupconsisting of a mammalian cell, an insect cell, a plant cell, a yeastcell and a protozoa cell.

According to still further features in the described preferredembodiments the cell is a bacterial cell.

According to yet an additional aspect of the present invention there isprovided an isolated preparation of bacterial derived inclusion bodiescomprising over 30 percent by weight of an immuno-molecule as describedherein

According to still an additional aspect of the present invention thereis provided a method of producing an immuno-molecule comprising:expressing, in bacteria, the immuno-molecule which comprises: a solublehuman MHC class I effector domain which includes a functional human β-2microglobulin and a functional human MHC class I heavy chain linkedthereto; and a targeting domain being linked to the soluble human MHCclass I effector domain; and isolating the immuno-molecule.

According to further features in preferred embodiments of the inventiondescribed below, immuno-molecule further comprises an MHC-restrictedpeptide linked to the functional human β-2 microglobulin, the methodfurther comprising refolding the immuno-molecule to thereby generate anMHC class I-MHC-restricted peptide complex.

According to still further features in the described preferredembodiments isolating the immuno-molecule is via size exclusionchromatography.

According to still further features in the described preferredembodiments an MHC-restricted peptide is co-expressed along with theimmuno-molecule in the bacteria.

According to still further features in the described preferredembodiments expressing, in the bacteria, the immuno-molecule is effectedsuch that the immuno-molecule forms inclusion bodies in the bacteria.

According to still further features in the described preferredembodiments the MHC-restricted peptide and the immuno-molecule co-forminclusion bodies in the bacteria.

According to still further features in the described preferredembodiments isolating the immuno-molecule further comprises: denaturingthe inclusion bodies so as to release protein molecules therefrom; andrenaturing the protein molecules.

According to still further features in the described preferredembodiments renaturing the protein molecules is effected in the presenceof an MHC-restricted peptide.

According to still further features in the described preferredembodiments the MHC-restricted peptide is co-expressed in the bacteria.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a new means with which tocombat cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-F demonstrate binding of in vitro refolded scHLA-A2 complexesto CTLs. Melanoma differentiation antigen gp100-specific CTL clonesR6C12 and R1E2 were reacted with in vitro refolded purified scHLA-A2tetramers containing the G9-209M epitope recognized by R6C12 CTLs andG9-280V peptide recognized by R1E2 CTLs. CTLs were stained withFITC-anti-CD8 (FIGS. 1A and 1D), with PE-labeled scHLA-A2/G9-209Mtetramers (FIGS. 1B and 1F) and with scHLA-A2/G9-280V tetramers (FIGS.1C and 1E). R6C12 and R1E2 CTLs were stained with the specific G9-209Mand G9-280V tetramer, respectively but not with control tetramer.

FIG. 1G is a schematic representation of a scHLA-A2 complex used in theexperiments described under FIGS. 1A-F.

FIG. 1H demonstrates the nucleic (SEQ ID NO:1) and amino (SEQ ID NO:2)acid sequences of the scHLA-A2 schematically illustrated in FIG. 1G.

FIGS. 2A-D demonstrate the design, expression, purification andbiochemical characterization of B2M-aTac(dsFv). FIG. 2A—TheB2M-aTac(dsFv) construct was generated by fusing a single-chain MHC toan antibody variable Fv fragment. In the single-chain HLA-A2 gene, thehuman α-2m was fused to the three extracellular domains of HLA-A2 via aflexible 15-amino acid long linker [(Gly₄-Ser)₃, i.e., GGGGSGGGGSGGGGS(SEQ ID NO:3), encoded by GGCGGAGGAGGGTCCGGTGGCGGAGG TTCAGGAGGCGGTGGATCG(SEQ ID NO:15)]. The same peptide linker was used to connect the scHLAgene to the antibody Fv fragment. The VL variable domain of the antibodywas fused to the C-terminus of the scHLA-A2 gene while the VH variabledomain was expressed separately. The two plasmids were expressed inseparate cultures and the solubilized, reduced inclusion bodies werecombined to form a disulfide-stabilized Fv fragments (dsFv) in which theFv variable domains are stabilized by interchain disulfide bondsengineered between conserved framework residues. FIG. 2B shows SDS-PAGEanalysis of the inclusion bodies from bacterial cultures induced toexpress the components of the B2M-aTac(dsFv); B2M-aTacVL and aTacVH.FIG. 3C shows SDS-PAGE analyses on non-reducing and reducing gels ofB2M-aTac(dsFv) after ion-exchange purification on Q-Sepharose column.FIG. 4D demonstrates binding of refolded B2M-aTac(dsFv)/G9-209M to thetarget antigen, p55. Detection of binding was with theconformational-specific MAb w6/32.

FIG. 2E demonstrates the nucleic (SEQ ID NO:4, linker sequence is shownin non-capital letters) and amino (SEQ ID NO:5) acid sequences of theB2M-aTacVL schematically illustrated in FIG. 2A as a part ofB2M-aTac(dsFv).

FIG. 2F demonstrates the nucleic (SEQ ID NO:6) and amino (SEQ ID NO:7)acid sequences of the aTacVH schematically illustrated in FIG. 2A as apart of B2M-aTac(dsFv).

FIGS. 3A-F demonstrate binding of B2M-aTac(dsFv) to HLA-A2-negativetumor target cells. Flow cytometry analysis of the binding ofB2M-aTac(dsFv) to antigen-positive HLA-A2-negative cells. FIG. 3A showbinding of anti-Tac Mab (red) to A431; FIG. 3B shows binding of anti-TacMAb to Tac (p55)-transfected A431 (ATAC4) cells (red); FIG. 3C showsbinding of anti-HLA-A2 MAb BB7.2 to A431 cells incubated (red) or not(blue) with B2M-aTac(dsFv); FIG. 3D shows binding of MAb BB7.2 top55-transfected ATAC4 cells preincubated (red) or not (blue) withB2M-aTac(dsFv); FIG. 3E shows binding of anti-Tac MAb (red) to leukemicHUT102W cells; and FIG. 3F shows binding of MAb BB7.2 to HUT102W cellspreincubated (red) or not (blue) with B2M-aTac(dsFv). In all cases,control cells with secondary antibody are shown in black.

FIGS. 4A-E demonstrate potentiation of CTL-mediated lysis ofHLA-A2-negative tumor cells by B2M-aTac(dsFv). Target cells coated ornot with the B2M-aTac(dsFv)-peptide complexes were incubated withmelanoma reactive gp100-peptide specific CTLs in a ³⁵-Methionine-releaseassay. FIG. 4A—A431 and p55-transfected ATAC4 HLA-A2⁻ cells werepreincubated or not with B2M-aTac(dsFv)/G9-209M complexes followed byincubation with the G9-209M-specific CTL, R6C12. Control are cellsincubated with medium alone; FIG. 4B—A431 and p55-transfected ATAC4HLA-A2⁻ cells were preincubated with B2M-aTac(dsFv)/G9-209M complexesfollowed by incubation with R6C12 CTLs. FM3D are HLA-A2⁺, gp100⁺melanoma cells; FIGS. 4C and 4D—p55-transfected ATAC4 cells werepreincubated with B2M-aTac(dsFv) complexes refolded with theHLA-A2-restricted peptides G9-209M, G9-280V, and TAX followed byincubation with the G9-209M-specific CTL clone R6C12 in FIG. 4C or theG9-280V-specific CTL clone R1E2 in FIG. 4D; FIG. 4E—HUT102W and CRII-2HLA-A2⁻ leukemic cells were preincubated (w) or not (w/o) withB2M-aTac(dsFv) complexes containing the appropriate peptide followed byincubation with the G9-209M-specific R6C12 CTLs or G9-280V-specific R1E2CTLs as indicated.

FIG. 5 is a plot demonstrating the results of an in vivo win assay withB2m-aTac(dsFv). ATAC4 cells (1×10⁵) were mixed with R6C12 CTL (1×10⁶)(E:T 10:1) in the presence or absence of B2M-aTac(dsFv) (20-50 g/ml) in200 l. The mixture was injected subcotaneously to nude mice and theappearance of tumors was observed. ATAC4 cells alone were used ascontrol.

FIG. 6 is a schematic illustration of preferred immuno-moleculesaccording to the present invention, wherein lines between boxesrepresent covalent linkage (e.g., translational fusion) between moietiesin the boxes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of (i) novel immuno molecules; (ii) methods ofpreparing same; (iii) nucleic acid constructs encoding same; and (iv)methods of using same for selective killing of cells, cancer cells inparticular.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Tumor progression is often associated with secretion of immunesuppressive factors and/or downregulation of MHC class I antigenpresentation functions (1-5, 14, 15). The inference is that tumors haveelaborated strategies to circumvent an apparently effective immuneresponse. Significant progress toward developing vaccines that canstimulate an immune response against tumors has involved theidentification of the protein antigens associated with a given tumortype and epitope mapping of tumor antigens for HLA class I and class IIrestricted binding motifs were identified and are currently being usedin various vaccination programs (6, 9, 11-13). MHC class I moleculespresenting the appropriate peptides are necessary to provide thespecific signals for recognition and killing by CTLs. However, theprinciple mechanism of tumor escape is the loss, downregulation oralteration of HLA profiles that may render the target cell unresponsiveto CTL lysis, even if the cell expresses the appropriate tumor antigen.In human tumors, HLA loss may be as high as 50%, suggesting that areduction in protein levels might offer a survival advantage to thetumor cells (14, 15).

The present invention presents a new approach to circumvent thisproblem. While reducing the present invention to practice,tumor-specific targeting of class I MHC-peptide complexes onto tumorcells was shown to be an effective and efficient strategy to renderHLA-A2-negative cells susceptible to lysis by relevant HLA-A2-restrictedCTLs. This new strategy of redirecting CTLs against tumor cells takesadvantage of the use of recombinant antibody fragments or ligands thatcan localize on malignant cells that express a tumor marker (antigen,e.g., receptor), usually associated with the transformed phenotype (suchas growth factor receptors, differentiation antigens), with a relativelyhigh degree of specificity. The tumor targeting recombinant antibodyfragments used while reducing the present invention to practice,constituted of the Fv variable domains which are the smallest functionalmodules of antibodies necessary to maintain antigen binding. This makesthem especially useful for clinical applications, not only forgenerating the molecule described herein but also for making otherantibody fusion proteins, such as recombinant Fv immunotoxins orrecombinant antibody-cytokine fusions (37, 38), because their small sizeimproves tumor penetration.

The antibody targeting fragment or targeting ligand is fused to asingle-chain HLA molecule that can be folded efficiently andfunctionally around an HLA-A2-restricted peptide. This approach can beexpanded to other major HLA alleles and many types of tumorspecificities which are dictated by the recombinant antibody fragments,thus, generating a new family of immunotherapeutic agents that may beused to augment and potentiate anti-tumor activities. Together with theapplication of monoclonal antibodies for cancer therapy this approachmay be regarded as a link between anti-tumor antibodies andcell-mediated immunotherapy.

Recombinant antibodies have been used already to redirect T cells usinga classical approach of bispecific antibodies in which one antibody armis directed against a tumor-specific antigen and the other arm againstan effector cell-associated molecule such as CD3 for CTLs and CD16 forNK cells (39).

Ligands that bind to tumor cells have also been used already to target avariety of toxins to tumor cells. See, for example, references 50-52with respect to EGF, TGF, IL-2 and IL-3.

A major advantage of the approach of the present invention is the use ofa recombinant molecule that can be produced in a homogeneous form andlarge quantities. Importantly, the size of the B2M-dsFv molecule atapproximately 65 kDa (generated with any antibody dsFv fragment) isoptimal with respect to the requirements needed for good tumorpenetration on one hand and relatively long half life and stability inthe circulation of the other (40). A recent study describing thegeneration of antibody-class I MHC tetramers was published in whichefficient CTL-mediated killing of tumor target cells was observed usingFab-streptavidin-MHC tetramer conjugates (41). The limitation of thisapproach, in comparison to the recombinant antibody fragment-monomericscMHC fusion described herein, is the large size of these molecules ofaround 400 kDa and the fact that soluble MHC tetramers can induce T cellactivation themselves whereas monomeric MHC molecule can not induceactivation unless in a relatively high local concentration (42-44).

The coating of tumor cells which had downregulated their own MHCexpression through the use of this targeting approach potentiates thecells for CTL-mediated killing while using a target on the tumor cellsthat is usually involved in the transformation process, most classicalexamples are growth factor receptors such as the IL-2R as used herein.This fact also favors the idea that using this approach escape mutantswhich down regulate the targeted receptor are not likely to have agrowth advantage because the receptor is directly involved in keysurvival functions of the cancer cells.

Another advantage to the antibody approach presented herein is the factthat these new agents can be designed around the desired peptidespecificity, namely the refolding of the B2M-Fv molecule can beperformed around any appropriate MHC-restricted peptide. In the Examplespresented herein, HLA-A2-restricted tumor-specific CTLs recognizing Tcell epitopes derived from the melanoma differentiation antigen gp100was employed. However, the kind of antigen-reactive CTL to be redirectedto kill the tumor cells can be defined by other antigenic peptides basedon recent knowledge of immune mechanisms in health and disease. Forexample, the identification of tumor-specific CTL responses in patientsmay suggest that these may be efficient to target. However, recentstudies have demonstrated that these tumor-specific CTLs are not alwaysoptimal since they are often present only at very low frequencies oreven when they are present at high frequencies they may be notfunctional or anergic (7). Thus, a more active and promising source ofCTLs can be recruited from circulating lymphocytes directed againstcommon and very immunogenic T cell epitopes such as derived from virusesor bacterial toxins which can also elicit a good memory response(45,46). It has been shown that CTL precursors directed againstinfluenza, EBV, CMV epitopes (peptides) are maintained in highfrequencies in the circulation of cancer patients as well as healthyindividuals and these CTLs are usually active and with a memoryphenotype (45, 46). Thus, these CTLs would be the source of choice to beredirected to the tumor cells through the use of a B2M-Fv moleculegenerated loaded with such viral-derived epitopes. The optimal agent isa B2M-Fv molecule in which the antigenic peptide is also covalentlylinked to the complex through the use of a flexible linker connectingthe peptide to the N-terminus of the β-2 microglobulin. This constructwill ensure optimal stability for the scMHC complex in vivo because thestabilizing peptide is connected covalently and can not leave easily theMHC peptide-binding groove. This type of single-chain peptide-MHCmolecules were generated previously in murine and human systems forvarious functional and structural studies (47, 48). An additional optionis to use antigenic peptide-derivatives that are modified at the“anchoring residues” in a way that increases their affinity to the HLAbinding groove (27).

There are also several options for the type of Fv fragment to be used asthe targeting moiety. In addition to the dsFv type of fragment, employedwhile reducing the present invention to practice, a single-chain Fvfragment (scFv) can be used in which the antibody VH and VL domains areconnected via a peptide linker. In such case the B2M-Fv molecule isencoded by one plasmid which avoids possible contamination withsingle-domain B2M molecules.

Another important aspect of the present invention which is supported byothers is the fact that the coating of antigenic MHC-peptide complexeson the surface of tumor cells without transmembrane anchoring issufficient to induce their efficient lysis by specific CTLs without theknowledge whether autologous accessory molecules of the target tumorcells are present at all and are playing a role in such CTL-mediatedkilling. This observation results from the fact that a local highconcentration of coated MHC-peptide complexes displaying one particularT cell epitope (peptide) is formed on the targeted cells which greatlyexceeds the natural density of such complexes displayed on the surfaceof cells. In the case of the IL-2R α subunit, several hundred tothousands sites per cell are present on the target cells, in comparisonto very few complexes containing one particular peptide expected to bepresent on cells, which may be sufficient for effective and efficientkilling even without the involvement of accessory molecules. This iswithout taking into consideration the downregulation of class I MHCexpression as an escape mechanism. Further indication for thispossibility is found through the findings that MHC tetramers can induceT cell activation by themselves (44) including the recent observationthat CTL activation by MHC tetramers without accessory molecules can bedemonstrated at the single cell level (Cohen, Denkberg, Reiter;manuscript submitted).

In conclusion, the results presented herein provide a cleardemonstration of the usefulness of the approach of the present inventionto recruit active CTLs for tumor cell killing via cancer-specificantibody or ligand guided targeting of scMHC-peptide complexes. Theseresults pave the way for the development of a new immunotherapeuticapproach based on naturally occurring cellular immune responses whichare redirected against the tumor cells.

According to one aspect of the present invention there is provided animmuno-molecule which comprises a soluble human MHC class I effectordomain; and a targeting domain, either antibody targeting domain orligand targeting domain, which is linked to the soluble human MHC classI effector domain. Preferably, the immuno-molecule has a molecularweight below 100 kDa. The soluble human MHC class I effector domain andthe targeting domain are preferably translationally fused, optionallywith a translationally fused peptide linker in-between. However, otherways to covalently link the soluble human MHC class I effector domainand the targeting domain are described hereinbelow.

FIG. 6 demonstrates several preferred immuno-molecules of the presentinvention, identified as (i)-(xiv). All of the molecules comprise asingle chain and soluble MHC, which includes functional human β-2microglobulin linked to functional human MHC class I heavy chain, whichpreferably comprises domains a 1-3. Preferably, the functional human β-2microglobulin and the functional human MHC class I heavy chain aretranslationally fused, optionally with a translationally fused peptidelinker in-between. However, as if further detailed below, the functionalhuman β-2 microglobulin and the functional human MHC class I heavy chaincan be covalently linked to one another in other ways.

As used herein the term “functional” when used in reference to the β-2microglobulin and heavy chain polypeptides of a single chain MHC class Icomplex refers to any portion of each which is capable of contributingto the assembly of a functional single chain MHC class I complex (i.e.,capable of binding and presenting to CTLs specific MHC-restrictedantigenic peptides when complexed).

The phrases “translationally fused” and “in frame” are interchangeablyused herein to refer to polypeptides encoded by polynucleotides whichare covalently linked to form a single continuous open reading framespanning the length of the coding sequences of the linkedpolynucleotides. Such polynucleotides can be covalently linked directlyor preferably indirectly through a spacer or linker region encoding alinker peptide.

Molecules (i)-(vi) and (xiii) further comprise a MHC-restricted peptidecovalently linked thereto. The MHC-restricted peptide is preferablylinked to the functional human β-2 microglobulin. Preferably, theMHC-restricted peptide and the functional human β-2 microglobulin aretranslationally fused, optionally with a translationally fused peptidelinker in-between. However, as if further detailed below, theMHC-restricted peptide and the functional human β-2 microglobulin can becovalently linked to one another in other ways.

Molecules (vii)-(xii) and (xiv) further comprise a MHC-restrictedpeptide which is not covalently linked thereto. In both cases, however,the MHC-restricted peptide is selected to complex with the functionalhuman MHC class I heavy chain upon refolding, as if further describedbelow.

The MHC-restricted peptide is preferably derived from a common pathogen,such as influenza, hepatitis, etc. The pathogen from which theMHC-restricted peptide is derived is selected according to severalcriteria as follows: (i) preferably, a large portion of the populationwas exposed to the pathogen or its antigens via infection ofvaccination; (ii) an active vaccination is available for the pathogen,so as to be able to boost the immune response; and (iii) relatively hightiter of CTLs with long term memory for the pathogen are retained ininfected or vaccinated patients.

In the alternative, the MHC peptide is derived from a tumor associatedor specific antigen. It was shown that MHC-restricted peptides derivedfrom tumor associated or specific antigen can be used to elicit anefficient CTL response. To this end, see, for example, WO 00/06723,which is incorporated herein by reference.

The targeting domain can be an antibody targeting domain (molecules(i)-(xii)) or a ligand targeting domain (molecules (xiii) and (xiv)).

According to a one preferred embodiment of the present invention theantibody targeting domain comprises a variable region of a light chainof an antibody linked to the effector domain (see molecules (i) and(vii) of FIG. 6). Preferably, the variable region of the light chain ofthe antibody and the effector domain are translationally fused,optionally with a translationally fused peptide linker in-between.However, other ways to covalently link the variable region of the lightchain of the antibody and the effector domain are described below.

According to another preferred embodiment, the antibody targeting domainfurther comprises a variable region of a heavy chain of an antibodylinked to the variable region of the light chain of the antibody (seemolecules (iii)-(vi) and (ix)-(xii) of FIG. 6). Preferably, the variableregion of the heavy chain of the antibody and the variable region of thelight chain of the antibody are translationally fused, optionally with atranslationally fused peptide linker in-between (see molecules (vi) and(x) of FIG. 6). However, other ways to covalently link the variableregion of the heavy chain of the antibody and the variable region of thelight chain of the antibody are disclosed herein.

For example, the variable region of the heavy chain of the antibody canbe linked to the variable region of the light chain of the antibody viaat least one S—S bond, generating a dsFV moiety (see, for example,molecules (v) and (xi) in FIG. 6)).

According to a another preferred embodiment of the present invention theantibody targeting domain comprises a variable region of a heavy chainof an antibody linked to the effector domain (see molecules (ii) and(viii) of FIG. 6). Preferably, the variable region of the heavy chain ofthe antibody and the effector domain are translationally fused,optionally with a translationally fused peptide linker in-between (seemolecules (iii) and (ix) of FIG. 6). However, other ways to covalentlylink the variable region of the heavy chain of the antibody and theeffector domain are described below.

According to another preferred embodiment, the antibody targeting domainfurther comprises a variable region of a light chain of an antibodylinked to the variable region of the heavy chain of the antibody (seemolecules (iii), (vi), (ix) and (xii) of FIG. 6). Preferably, thevariable region of the light chain of the antibody and the variableregion of the heavy chain of the antibody are translationally fused,optionally with a translationally fused peptide linker in-between (seemolecules (iii) and (ix) of FIG. 6). However, other ways to covalentlylink the variable region of the light chain of the antibody and thevariable region of the heavy chain of the antibody are disclosed herein.

For example, the variable region of the light chain of the antibody canbe linked to the variable region of the heavy chain of the antibody viaat least one S—S bond, generating a dsFV moiety (see, for example,molecules (vi) and (xii) in FIG. 6)).

The antibody targeting domain in the molecule of the invention isselected capable of binding to a tumor associated or specific antigen.It will be appreciated in this respect that presently there are severalhundred identified tumor associated or specific antigens, associatedwith various solid and non solid tumors, and further that monoclonalantibodies were developed for many of which. In other words, the aminoacid and nucleic acid sequences of many antibodies which specificallybind to tumor associated or specific antigens is either already known orcan be readily determined by analyzing the hybridomas producing suchantibodies.

The molecules described in FIG. 6 are composed of a single polypeptide[e.g., molecules (i)-(iv) and (xiii)], two polypeptides [molecules (v),(vi), (vi)-(x) and (xiv)] or three polypeptides [molecules (xi) and(xii)].

The terms peptide and polypeptide are used herein interchangeably. Eachof the polypeptides can be synthesized using any method known in theart. Hence, it will be appreciated that the immuno-molecules of thepresent invention or portions thereof can be prepared by several ways,including solid phase protein synthesis, however, in the preferredembodiment of the invention, at least major portions of the molecules,e.g., the soluble human MHC class I effector domain (with or without theMHC-restricted peptide) and the antibody targeting domain (as a scFV oras an arm of a dsFv) are generated by translation of a respectivenucleic acid construct or constructs.

So, one to three open reading frames are required to synthesize themolecules of FIG. 6 via translation. These open reading frames canreside on a single, two or three nucleic acid molecules. Thus, forexample, a single nucleic acid construct can carry all one, two or threeopen reading frames. One to three cis acting regulatory sequences can beused to control the expression of the one to three open reading frames.For example, a single cis acting regulatory sequence can control theexpression of one, two or three open reading frames, in a cistrone-likemanner. In the alternative, three independent cis acting regulatorysequences can be used to control the expression of the three openreading frames. Other combinations are also envisaged.

In cases where the MHC-restricted peptide is not covalently linked tothe remaining portions of the molecule (see in FIG. 6 molecules(vii)-(xii)), it is preferably prepared via solid phase techniques, asit is generally a short peptide of less than 10 amino acids.

The open reading frames and the cis acting regulatory sequences can becarried by one to three nucleic acid molecules. For example, each openreading frame and its cis acting regulatory sequence are carried by adifferent nucleic acid molecule, or all of the open reading frames andtheir associated cis acting regulatory sequences are carried by a singlenucleic acid molecule. Other combinations are also envisaged.

Expression of the polypeptide(s) can be effected bytransformation/transfection and/or co-transformation/co-transfection ofa single cell or a plurality of cells with any of the nucleic acidmolecules, serving as transformation/transfection vectors (e.g., asplasmids, phages, phagemids or viruses).

Hence, according to another aspect of the present invention there isprovided a nucleic acid construct encoding an immuno-molecule. Theconstruct according to this aspect of the invention comprises a firstpolynucleotide encoding a soluble human MHC class I effector domain; anda second polynucleotide encoding a targeting domain, either an antibodytargeting domain or a ligand targeting domain. The first polynucleotideand the second polynucleotide are selected and being joined togethersuch that the soluble human MHC class I effector domain and thetargeting domain are translationally fused, optionally via a peptidelinker in-between.

According to still another aspect of the present invention there isprovided a nucleic acid construct encoding an immuno-molecule. Theconstruct according to this aspect of the invention comprises a firstpolynucleotide encoding a soluble human MHC class I effector domain; anda second polynucleotide encoding a variable region of one of a lightchain or a heavy chain of an antibody targeting domain. The firstpolynucleotide and the second polynucleotide are selected and beingjoined together such that the soluble human MHC class I effector domainand the variable region of the one of the light chain and heavy chain ofthe antibody targeting domain are translationally fused optionally via apeptide linker in-between. The construct according to this aspect of theinvention further comprises and a third polynucleotide encoding theother of the one of the light chain and heavy chain of the antibodytargeting domain. The third polynucleotide may be selected so as toencode a separate polypeptide, so as to allow generation of a dsFV, orto encode a polypeptide which is translationally fused to the secondnucleic acid, so as to allow generation of a scFV.

According to an additional aspect of the present invention there isprovided a nucleic acid construct system. The construct system comprisesa first nucleic acid construct which comprises a first polynucleotideencoding a soluble human MHC class I effector domain; and a secondpolynucleotide encoding a variable region of one of a light chain or aheavy chain of an antibody targeting domain. The first polynucleotideand the second polynucleotide are selected and being joined togethersuch that the soluble human MHC class I effector domain and the variableregion of the one of the light chain and heavy chain of the antibodytargeting domain are translationally fused optionally via a peptidelinker in-between. The construct system further comprises a secondnucleic acid construct which comprises a third polynucleotide encodingthe other of the one of the light chain and heavy chain of the antibodytargeting domain. These constructs may be cointroduced into the samecell or into different cells. In the first case, the constructs makingthe construct system may be mixed together, whereas in the second case,the constructs making the construct system are kept unmixed in separatecontainers.

Whenever and wherever used, the linker peptide is selected of an aminoacid sequence which is inherently flexible, such that the polypeptidesconnected thereby independently and natively fold following expressionthereof, thus facilitating the formation of a functional single chain(sc) human MHC class I complex, targeting scFv or ligand and/or humanMHC class I-MHC restricted antigen complex.

Any of the nucleic acid constructs described herein comprise at leastone cis acting regulatory sequence operably linked to the codingpolynucleotides therein. Preferably, the cis acting regulatory sequenceis functional in bacteria. Alternatively, the cis acting regulatorysequence is functional in yeast. Still alternatively, the cis actingregulatory sequence is functional in animal cells. Yet alternatively,the cis acting regulatory sequence is functional in plant cells.

The cis acting regulatory sequence can include a promoter sequence andadditional transcriptional or a translational enhancer sequences all ofwhich serve for facilitating the expression of the polynucleotides whenintroduced into a host cell. Specific examples of promoters aredescribed hereinbelow in context of various eukaryotic and prokaryoticexpression systems and in the Examples section which follows.

It will be appreciated that a single cis acting regulatory sequence canbe utilized in a nucleic acid construct to direct transcription of asingle transcript which includes one or more open reading frames. In thelater case, an internal ribosome entry site (IRES) can be utilized so asto allow translation of the internally positioned nucleic acid sequence.

According to another aspect of the present invention there is provided atransformed cell which comprises any one or more of the nucleic acidconstructs or the nucleic acid construct system described herein. Thecell, according to this aspect of the invention can be a eukaryotic cellselected from the group consisting of a mammalian cell, an insect cell,a plant cell, a yeast cell and a protozoa cell, or it can be a bacterialcell.

Whenever co-expression of independent polypeptides in a single cell isof choice, the construct or constructs employed must be configured suchthat the levels of expression of the independent polypeptides areoptimized, so as to obtain highest proportions of the final product.

Preferably a promoter (being an example of a cis acting regulatorysequence) utilized by the nucleic acid construct(s) of the presentinvention is a strong constitutive promoter such that high levels ofexpression are attained for the polynucleotides following host celltransformation.

It will be appreciated that high levels of expression can also beeffected by transforming the host cell with a high copy number of thenucleic acid construct(s), or by utilizing cis acting sequences whichstabilize the resultant transcript and as such decrease the degradationor “turn-over” of such a transcript.

As used herein, the phrase “transformed cell” describes a cell intowhich an exogenous nucleic acid sequence is introduced to thereby stablyor transiently genetically alter the host cell. It may occur undernatural or artificial conditions using various methods well known in theart some of which are described in detail hereinbelow in context withspecific examples of host cells.

The transformed host cell can be a eukaryotic cell, such as, forexample, a mammalian cell, an insect cell, a plant cell, a yeast celland a protozoa cell, or alternatively, the cell can be a bacterial cell.

When utilized for eukaryotic host cell expression, the nucleic acidconstruct(s) according to the present invention can be a shuttle vector,which can propagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for expression in eukaryotic host cells. The nucleic acidconstruct(s) according to the present invention can be, for example, aplasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or anartificial chromosome.

According to another preferred embodiment of the present invention thehost cell is a mammalian cell of, for example, a mammalian cell culture.Suitable mammalian expression systems include, but are not limited to,pcDNA3, pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto,pCMV/myc/cyto, pCR3.1, which are available from Invitrogen, pCI which isavailable from Promega, pBK-RSV and pBK-CMV which are available fromStratagene, pTRES which is available from Clontech, and theirderivatives.

Insect cell cultures can also be utilized to express the nucleic acidsequences of the present invention. Suitable insect expression systemsinclude, but are not limited to the baculovirus expression system andits derivatives which are commercially available from numerous supplierssuch as Invitrogen (maxBac™) Clontech (BacPak™), or Gibco (Bac-to-Bac™).

Expression of the nucleic acid sequences of the present invention canalso be effected in plants cells. As used herein, the phrase “plantcell” can refer to plant protoplasts, cells of a plant tissue culture,cells of plant derived tissues or cells of whole plants.

There are various methods of introducing nucleic acid constructs intoplant cells. Such methods rely on either stable integration of thenucleic acid construct or a portion thereof into the genome of the plantcell, or on transient expression of the nucleic acid construct in whichcase these sequences are not stably integrated into the genome of theplant cell.

There are two principle methods of effecting stable genomic integrationof exogenous nucleic acid sequences such as those included within thenucleic acid construct of the present invention into plant cell genomes:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; or by the direct incubation of DNA with germinatingpollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds.Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London,(1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986)83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure, see for example, Horsch et al. in PlantMolecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht(1988) p. 1-9. A supplementary approach employs the Agrobacteriumdelivery system in combination with vacuum infiltration. TheAgrobacterium system is especially viable in the creation of stablytransformed dicotyledenous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, protoplasts are briefly exposed to a strong electricfield. In microinjection, the DNA is mechanically injected directly intothe cells using very small micropipettes. In microparticle bombardment,the DNA is adsorbed on microprojectiles such as magnesium sulfatecrystals, tungsten particles or gold particles, and the microprojectilesare physically accelerated into cells or plant tissues. Direct DNAtransfer can also be utilized to transiently transform plant cells.

In any case suitable plant promoters which can be utilized for plantcell expression of the first and second nucleic acid sequences, include,but are not limited to CaMV 35S promoter, ubiquitin promoter, and otherstrong promoters which can express the nucleic acid sequences in aconstitutive or tissue specific manner.

Plant viruses can also be used as transformation vectors. Viruses thathave been shown to be useful for the transformation of plant cell hostsinclude CaV, TMV and BV. Transformation of plants using plant viruses isdescribed in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), JapanesePublished Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667(BV); and Gluzman, Y. et al., Communications in Molecular Biology: ViralVectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988).Pseudovirus particles for use in expressing foreign DNA in many hosts,including plants, is described in WO 87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, the constructions can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe nucleic acid sequences described above. The virus can then beexcised from the plasmid. If the virus is a DNA virus, a bacterialorigin of replication can be attached to the viral DNA, which is thenreplicated by the bacteria. Transcription and translation of this DNAwill produce the coat protein which will encapsidate the viral DNA. Ifthe virus is an RNA virus, the virus is generally cloned as a cDNA andinserted into a plasmid. The plasmid is then used to make all of theconstructions. The RNA virus is then produced by transcribing the viralsequence of the plasmid and translation of the viral genes to producethe coat protein(s) which encapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

Yeast cells can also be utilized as host cells by the present invention.Numerous examples of yeast expression vectors suitable for expression ofthe nucleic acid sequences of the present invention in yeast are knownin the art and are commercially available. Such vectors are usuallyintroduced in a yeast host cell via chemical or electroporationtransformation methods well known in the art. Commercially availablesystems include, for example, the pYES™ (Invitrogen) or the YEX™(Clontech) expression systems.

It will be appreciated that when expressed in eukaryotic expressionsystems such as those described above, the nucleic acid constructpreferably includes a signal peptide encoding sequence such that thepolypeptides produced from the first and second nucleic acid sequencesare directed via the attached signal peptide into secretion pathways.For example, in mammalian, insect and yeast host cells, the expressedpolypeptides can be secreted to the growth medium, while in plantexpression systems the polypeptides can be secreted into the apoplast,or directed into a subcellular organelle.

According to a presently most preferred embodiment of the invention, thehost cell is a bacterial cell, such as, for example, E. coli. Abacterial host can be transformed with the nucleic acid sequence viatransformation methods well known in the art, including for example,chemical transformation (e.g., CaCl₂) or electroporation.

Numerous examples of bacterial expression systems which can be utilizedto express the nucleic acid sequences of the present invention are knownin the art. Commercially available bacterial expression systems include,but are not limited to, the pET™ expression system (Novagen), pSE™expression system (Invitrogen) or the pGEX™ expression system(Amersham).

As is further described in the Examples section which follows, bacterialexpression is particularly advantageous since the expressed polypeptidesform substantially pure inclusion bodies readily amenable to recoveryand purification of the expressed polypeptide.

Thus, according to yet another aspect of the present invention there isprovided a preparation of bacterial derived inclusion bodies which arecomposed of over 30 percent, preferably over 50%, more preferably over75%, most preferably over 90% by weight of the recombinant polypeptideor a mixture of polypeptides of the present invention. The isolation ofsuch inclusion bodies and the purification of the polypeptide(s)therefrom are described in detail in the Examples section which follows.

As demonstrated in the Examples section that follows, bacterialexpression of the polypeptide(s) can provide high quantities of pure andfunctional immunomolecules.

According to an additional aspect of the present invention there isprovided a method of producing an immunomolecule of the invention. Themethod according to this aspect of the present invention utilizes any ofthe nucleic acid construct(s) described for expressing, in bacteria, athe polypeptide(s) described herein.

Following expression, the polypeptide(s) is/are isolated and purified asdescribed below.

As is further described in the Examples section which follows, theexpressed polypeptide(s) form substantially pure inclusion bodies whichare readily isolated via fractionation techniques well known in the artand purified via for example denaturing-renaturing steps.

Preferably, the polypeptide(s) of the invention are renatured andrefolded in the presence of a MHC-restricted peptide, which is eitherlinked to, co-expressed with or mixed with other polypeptides of theinvention and being capable of binding the single chain MHC class Ipolypeptide. As is further described in the Examples section thisenables to generate a substantially pure MHC class I-antigenic peptidecomplex which can further be purified via size exclusion chromatography.

It will be appreciated that the MHC-restricted peptide used forrefolding can be co-expressed along with (as an independent peptide) orbe fused to the soluble human MHC class I polypeptide in the bacteria.In such a case the expressed polypeptide and peptide co-form inclusionbodies which can be isolated and utilized for MHC class I-antigenicpeptide complex formation.

According to a further aspect of the present invention there is provideda method of selectively killing a cell in a patient, the cell presentingan antigen (e.g., a receptor). The method according to this aspect ofthe invention comprises administering to the patient an immuno-moleculewhich comprises: a soluble human MHC class I effector domain complexedwith an MHC-restricted peptide; and a targeting domain, either antibodyor ligand targeting domain, being linked to the soluble human MHC classI effector domain. The targeting domain serves for selectively bindingto the antigen; whereby, the soluble human MHC class I effector domaincomplexed with the MHC-restricted peptide initiates a CTL mediatedimmune response against the cell, thereby selectively killing the cellin vivo. The cell to be killed can be a cancer cell, in which case, thetargeting domain will be selected binding to a tumor associated antigencharacterized for said cancer cell.

The following sections provide specific examples and alternatives foreach of the various aspects of the invention described herein. Theseexamples and alternatives should not be regarded as limiting in any way,as the invention can be practiced in similar, yet somewhat differentways. These examples, however, teach one of ordinary skills in the arthow to practice various alternatives and embodiments of the invention.

Antibody:

The term “antibody” and the phrase “antibody targeting domain” as usedto describe this invention includes intact molecules as well asfunctional fragments thereof, such as Fab, F(ab′)₂, Fv and scFv that arecapable of specific, high affinity binding to an antigen. Thesefunctional antibody fragments are defined as follows: (i) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule, can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (ii) Fab′, the fragment of an antibody molecule that can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (iii) F(ab′)₂, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)₂ is a dimerof two Fab′ fragments held together by two disulfide bonds; (iv) Fv,defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (c) scFv or “single chain antibody”(“SCA”), a genetically engineered molecule containing the variableregion of the light chain and the variable region of the heavy chain,linked by a suitable polypeptide linker as a genetically fused singlechain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959.Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence.

The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

It will be appreciated that once the CDRs of an antibody are identified,using conventional genetic engineering techniques can be used to deviseexpressible polynucleotides encoding any of the forms or fragments ofantibodies described herein.

Ligand

The Table below provides non exhaustive examples of receptorsselectively expressed by a variety of tumor cells, their ligands andsequence information pertaining to the ligands, which sequenceinformation can be used in the construction of constructs andimmuno-molecules according to the present invention:

Genebank Genebank Accession No. Accession No. (Nucleic acid (Amino acidReceptor Tumor (Ref) Ligand sequence) Sequence) EGFR Breast, Brain, EGFL17029 AAB32226 Lung (Niv et al Curr. Pharm. Biotech. 2: 19-46, 2002)PDGFR Ovary, Breast PDGF XO6374 CAA29677 Mutant Liver, Brain EGF S51343AAB19486 EGFR IL-4R Renal IL-4 M13982 AAA59149 IL-6R Myeloma IL-6 M14584AAA59149 IL-10R Leukemias IL-10 M57627 AAA63207 EGFR Breast, Ovary, TGFM31172 AAA61157 VEGFR Carcinomas blood VEGF M32977 AAA35789 vessels KDRCarcinomas blood VEGF M32977 AAA35789 vessels

A Human Major Histocompatibility Complex (MHC) Class I:

The major histocompatibility complex (MHC) is a complex of antigensencoded by a group of linked loci, which are collectively termed H-2 inthe mouse and HLA in humans. The two principal classes of the MHCantigens, class I and class II, each comprise a set of cell surfaceglycoproteins which play a role in determining tissue type andtransplant compatibility. In transplantation reactions, cytotoxicT-cells (CTLs) respond mainly against foreign class I glycoproteins,while helper T-cells respond mainly against foreign class IIglycoproteins.

Major histocompatibility complex (MHC) class I molecules are expressedon the surface of nearly all cells. These molecules function inpresenting peptides which are mainly derived from endogenouslysynthesized proteins to CD8+ T cells via an interaction with the αβT-cell receptor. The class I MHC molecule is a heterodimer composed of a46-kDa heavy chain which is non-covalently associated with the 12-kDalight chain β-2 microglobulin. In humans, there are several MHChaplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24,HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, theirsequences can be found at the kabat database of Sequences of Proteins ofImmunological Interest which is incorporated herein by reference.

Peptides that Bind to Class I MHC Molecules; MHC-Restricted Antigens:

Class I, MHC-restricted peptides (also referred to hereininterchangeably as MHC-restricted antigens, HLA-restricted peptides,HLA-restricted antigens) which are typically 8-10-amino acid-long, bindto the heavy chain α1-α2 groove via two or three anchor residues thatinteract with corresponding binding pockets in the MHC molecule. The β-2microglobulin chain plays an important role in MHC class I intracellulartransport, peptide binding, and conformational stability. For most classI molecules, the formation of a heterodimer consisting of the MHC classI heavy chain, peptide (self or antigenic) and β-2 microglobulin isrequired for biosynthetic maturation and cell-surface expression.

Research studies performed on peptide binding to class I MHC moleculesenable to define specific MHC motifs functional in displaying peptidesderived from viral, tumor and self antigens that are potentiallyimmunogenic and might elicit specific response from cytotoxic Tlymphocytes (CTLs).

As used herein the term “peptide” refers to native peptides (eitherdegradation products or synthetically synthesized peptides) and furtherto peptidomimetics, such as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body, or more immunogenic. Suchmodifications include, but are not limited to, cyclization, N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O,CH₂—CH₂, S═C—NH, CH═CH or CF═CH, backbone modification and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified in Quantitative Drug Design, C.A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein. Further detailin this respect are provided hereinunder.

As used herein in the specification and in the claims section below theterm “amino acid” is understood to include the 20 naturally occurringamino acids; those amino acids often modified post-translationally invivo, including for example hydroxyproline, phosphoserine andphosphothreonine; and other unusual amino acids including, but notlimited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,nor-leucine and ornithine. Furthermore, the term “amino acid” includesboth D- and L-amino acids. Further elaboration of the possible aminoacids usable according to the present invention and examples ofnon-natural amino acids useful in MHC-1HLA-A2 recognizable peptideantigens are given hereinunder.

Based on accumulated experimental data, it is nowadays possible topredict which of the peptides of a protein will bind to MHC, class I.The HLA-A2 MHC class I has been so far characterized better than otherHLA haplotypes, yet predictive and/or sporadic data is available for allother haplotypes.

With respect to HLA-A2 binding peptides, assume the following positions(P1-P9) in a 9-mer peptide:

-   -   P1-P2-P3-P4-P5-P6-P7-P8-P9

The P2 and P2 positions include the anchor residues which are the mainresidues participating in binding to MHC molecules. Amino acid residesengaging positions P2 and P9 are hydrophilic aliphatic non-chargednatural amino (examples being Ala, Val, Leu, Ile, Gln, Thr, Ser, Cys,preferably Val and Leu) or of a non-natural hydrophilic aliphaticnon-charged amino acid (examples being norleucine (Nle), norvaline(Nva), α-aminobutyric acid). Positions P1 and P3 are also known toinclude amino acid residues which participate or assist in binding toMHC molecules, however, these positions can include any amino acids,natural or non-natural. The other positions are engaged by amino acidresidues which typically do not participate in binding, rather theseamino acids are presented to the immune cells. Further details relatingto the binding of peptides to MHC molecules can be found in Parker, K.C., Bednarek, M. A., Coligan, J. E., Scheme for ranking potential HLA-A2binding peptides based on independent binding of individual peptideside-chains. J Immunol. 152, 163-175, 1994, see Table V, in particular.Hence, scoring of HLA-A2.1 binding peptides can be performed using theHLA Peptide Binding Predictions software. This software is based onaccumulated data and scores every possible peptide in an analyzedprotein for possible binding to MHC HLA-A2.1 according to thecontribution of every amino acid in the peptide. Theoretical bindingscores represent calculated half-life of the HLA-A2.1-peptide complex.

Hydrophilic aliphatic natural amino acids at P2 and P9 can besubstituted by synthetic amino acids, preferably Nleu, Nval and/orα-aminobutyric acid. P9 can be also substituted by aliphatic amino acidsof the general formula —HN(CH₂)_(n)COOH, wherein n=3-5, as well as bybranched derivatives thereof, such as, but not limited to,

wherein R is, for example, methyl, ethyl or propyl, located at any oneor more of the n carbons.

The amino terminal residue (position P1) can be substituted bypositively charged aliphatic carboxylic acids, such as, but not limitedto, H₂N(CH₂)_(n)COOH, wherein n=2-4 and H₂N—C(NH)—NH(CH₂)_(n)COOH,wherein n=2-3, as well as by hydroxy Lysine, N-methyl Lysine orornithine (Orn). Additionally, the amino terminal residue can besubstituted by enlarged aromatic residues, such as, but not limited to,H₂N—(C₆H₆)—CH₂—COOH, p-aminophenyl alanine,H₂N—F(NH)—NH—(C₆H₆)—CH₂—COOH, p-guanidinophenyl alanine orpyridinoalanine (Pal). These latter residues may form hydrogen bondingwith the OH⁻ moieties of the Tyrosine residues at the MHC-1 N-terminalbinding pocket, as well as to create, at the same time aromatic-aromaticinteractions.

Derivatization of amino acid residues at positions P4-P8, should theseresidues have a side-chain, such as, OH, SH or NH₂, like Ser, Tyr, Lys,Cys or Orn, can be by alkyl, aryl, alkanoyl or aroyl. In addition, OHgroups at these positions may also be derivatized by phosphorylationand/or glycosylation. These derivatizations have been shown in somecases to enhance the binding to the T cell receptor.

Longer derivatives in which the second anchor amino acid is at positionP10 may include at P9 most L amino acids. In some cases shorterderivatives are also applicable, in which the C terminal acid serves asthe second anchor residue.

Cyclic amino acid derivatives can engage position P4-P8, preferablypositions P6 and P7. Cyclization can be obtained through amide bondformation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric(Dab) acid, di-aminopropionic (Dap) acid at various positions in thechain (—CO—NH or —NH—CO bonds). Backbone to backbone cyclization canalso be obtained through incorporation of modified amino acids of theformulas H—N((CH₂)_(n)—COOH)—C(R)H—COOH orH—N((CH₂)_(n)—COOH)—C(R)H—NH₂, wherein n=1-4, and further wherein R isany natural or non-natural side chain of an amino acid.

Cyclization via formation of S—S bonds through incorporation of two Cysresidues is also possible. Additional side-chain to side chaincyclization can be obtained via formation of an interaction bond of theformula —(—CH₂—)_(n)—S—CH₂—C—, wherein n=1 or 2, which is possible, forexample, through incorporation of Cys or homoCys and reaction of itsfree SH group with, e.g., bromoacetylated Lys, Orn, Dab or Dap.

Peptide bonds (—CO—NH—) within the peptide may be substituted byN-methylated bonds (—N(CH₃)—CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—),ketomethylen bonds (—CO—CH₂—), α-aza bonds (—NH—N(R)—CO—), wherein R isany alkyl, e.g., methyl, carba bonds (—CH₂—NH—), hydroxyethylene bonds(—CH(OH)—CH₂—), thioamide bonds (—CS—NH—), olefinic double bonds(—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH₂—CO—), wherein R is the “normal” side chain, naturallypresented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time. Preferably, but not inall cases necessary, these modifications should exclude anchor aminoacids.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

Tumor MHC-Restricted Antigens:

The references recited in the following Table provide examples of humanMHC class I, tumor MHC-restricted peptides derived from tumor associatedantigens (TAA) or protein markers associated with various cancers.Additional tumor MHC-restricted peptides derived from tumor associatedantigens (TAA) can be found on the BMI Biomedical Informatics Heidelbergwebsite.

Cancer TAA/Marker HLA Reference Transitional cell Uroplakin II HLA-A2 WO00/06723 carcinoma Transitional cell Uroplakin Ia HLA-A2 WO 00/06723carcinoma Carcinoma of the prostate specific HLA-A2 WO 00/06723 prostateantigen Carcinoma of the prostate specific HLA-A2 WO 00/06723 prostatemembrane antigen Carcinoma of the prostate acid HLA-A2 WO 00/06723prostate phosphatase Breast cancer BA-46 HLA-A2 WO 00/06723 Breastcancer Muc-1 HLA-A2 WO 00/06723 Melanoma Gp100 HLA-A2 Reference 54Melanoma MART1 HLA-A2 Reference 54 All tumors Telomerase HLA-A2Reference 54 Leukemia TAX HLA-A2 Reference 54 Carcinomas NY-ESO HLA-A2Reference 54 Melanoma MAGE-A1 HLA-A2 Reference 54 Melanoma MAGE-A3HLA-A24 Reference 54 Carcinomas HER2 HLA-A2 Reference 54 MelanomaBeta-catenine HLA-A24 Reference 54 Melanoma Tyrosinase HLA-DRB1Reference 54 Leukemia Bcr-abl HLA-A2 Reference 54 Head and neck Caspase8 HLA-B35 Reference 54

Viral MHC-Restricted Antigens:

The references recited in the following Table provide examples of humanMHC class I, viral MHC-restricted peptides derived from viral antigens.

Disease Viral antigen HLA AIDS (HTLV-1) HIV-1 RT 476-484 HLA-A2Influenza G I L G F V F T L HLA-A2 (SEQ ID NO: 16) CMV disease CMVHLA-A2 Burkitts Lymphoma TAX HLA-A2 Hepatitis C HCV HLA-A2 Hepatitis BHBV pre-S protein HLA-A2 85-66 S T N R Q S G R Q (SEQ ID NO: 17)HTLV-1 Leukemia HTLV-1 tax 11-19 HLA-A2 Hepatitis HBV surface HLA-A2antigen 185-194

Autoimmune MHC-Restricted Antigens:

The BMI Biomedical Informatics Heidelberg website provides examples ofhuman MHC class I, autoimmune MHC-restricted peptides derived fromautoimmune antigens.

Soluble MHC Class I Molecules:

Sequences encoding recombinant MHC class I and class II complexes whichare soluble and which can be produced in large quantities are describedin, for example, references 23, 24 and 41-53 and further in U.S. patentapplication Ser. No. 09/534,966 and PCT/IL01/00260 (published as WO01/72768), all of which are incorporated herein by reference. SolubleMHC class I molecules are available or can be produced for any of theMHC haplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24,HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8,following, for example the teachings of PCT/IL01/00260, as theirsequences are known and can be found at the kabbat data base, thecontents of the site is incorporated herein by reference. Such solubleMHC class I molecules can be loaded with suitable MHC-restrictedantigens and used for vaccination of Non-human mammal having cellsexpressing the human major histocompatibility complex (MHC) class I asis further detailed hereinbelow.

Chemical Conjugates:

Many methods are known in the art to conjugate or fuse (couple)molecules of different types, including peptides or polypeptides. Thesemethods can be used according to the present invention to couple asoluble human MHC class I effector domain with an antibody targetingdomain and optionally with an MHC-restricted antigen.

Two isolated peptides can be conjugated or fused using any conjugationmethod known to one skilled in the art. One peptide can be conjugated toanother using a 3-(2-pyridyldithio)propionic acid Nhydroxysuccinimideester (also called N-succinimidyl 3-(2-pyridyldithio)propionate)(“SDPD”) (Sigma, Cat. No. P-3415), a glutaraldehyde conjugationprocedure or a carbodiimide conjugation procedure.

SPDP Conjugation:

Any SPDP conjugation method known to those skilled in the art can beused. For example, in one illustrative embodiment, a modification of themethod of Cumber et al. (1985, Methods of Enzymology 112: 207-224) asdescribed below, is used.

A peptide (1.7 mg/ml) is mixed with a 10-fold excess of SPDP (50 mM inethanol) and the antibody is mixed with a 25-fold excess of SPDP in 20mM sodium phosphate, 0.10 M NaCl pH 7.2 and each of the reactionsincubated, e.g., for 3 hours at room temperature. The reactions are thendialyzed against PBS.

The peptide is reduced, e.g., with 50 mM DTT for 1 hour at roomtemperature. The reduced peptide is desalted by equilibration on G-25column (up to 5% sample/column volume) with 50 mM KH₂PO₄ pH 6.5. Thereduced peptide is combined with the SPDP-antibody in a molar ratio of1:10 antibody:peptide and incubated at 4° C. overnight to form apeptide-antibody conjugate.

Glutaraldehyde Conjugation:

Conjugation of a peptide with another peptide can be accomplished bymethods known to those skilled in the art using glutaraldehyde. Forexample, in one illustrative embodiment, the method of conjugation by G.T. Hermanson (1996, “Antibody Modification and Conjugation, inBioconjugate Techniques, Academic Press, San Diego) described below, isused.

The peptides (1.1 mg/ml) are mixed at a 10-fold excess with 0.05%glutaraldehyde in 0.1 M phosphate, 0.15 M NaCl pH 6.8, and allowed toreact for 2 hours at room temperature. 0.01 M lysine can be added toblock excess sites. After-the reaction, the excess glutaraldehyde isremoved using a G-25 column equilibrated with PBS (10% v/v sample/columnvolumes)

Carbodiimide Conjugation:

Conjugation of a peptide with another peptide can be accomplished bymethods known to those skilled in the art using a dehydrating agent suchas a carbodiimide. Most preferably the carbodiimide is used in thepresence of 4-dimethyl aminopyridine. As is well known to those skilledin the art, carbodiimide conjugation can be used to form a covalent bondbetween a carboxyl group of peptide and an hydroxyl group of one peptide(resulting in the formation of an ester bond), or an amino group of theone peptide (resulting in the formation of an amide bond) or asulfhydryl group of the one peptide (resulting in the formation of athioester bond).

Likewise, carbodiimide coupling can be used to form analogous covalentbonds between a carbon group of one peptide and an hydroxyl, amino orsulfhydryl group of the other peptide. See, generally, J. March,Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp.349-50 & 372-74 (3d ed.), 1985. By means of illustration, and notlimitation, the peptide is conjugated to another via a covalent bondusing a carbodiimide, such as dicyclohexylcarbodiimide. See generally,the methods of conjugation by B. Neises et al. (1978, Angew Chem., Int.Ed. Engl. 17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); E.P. Boden et al. (1986, J. Org. Chem. 50:2394) and L. J. Mathias (1979,Synthesis 561).

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Examples

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Experimental Methods

Peptides:

Peptides were synthesized by standard fluorenylmethoxycarbonyl chemistryand purified to >95% by reverse phase HPLC. The tumor associatedHLA-A2-restricted peptides used are: G9-209-2M (IMDQVPFSV, SEQ ID NO:8)and G9-280-9V (YLEPGPVTV, SEQ ID NO:9), both derived from the melanomadifferentiation antigen gp100 and are common immunodominant epitopes(32-34). These peptides are modified at the MHC anchor positions 2 (inG9-209-2M) and 9 (in G9-280-9V) to improve the binding affinity toHLA-A2 (27). The HTLV-1-derived peptide (LLFGYPVYV, SEQ ID NO:10) wasused as control.

Cell Lines:

A431, ATAC4 (epidermoid carcinoma), HUT102W and CRII-2 (leukemia, ATL)cells were maintained in RPMI+10% FCS. ATAC4 cells are human epidemoidcarcinoma A431 cells stably transfected with the IL-2 receptor α subunit(p55, Tac, CD25) (53). The transfected cells were maintained in growthmedium containing 500 μg/ml G418 (Gibco-BRL).

Plasmid Constructions:

The scMHC molecule was constructed as previously described by linkinghuman β2-microglobulin with the three extracellular domains of theHLA-A2 gene (24, 25, WO 01/72768). The VL(cys) and VH(cys) variabledomain genes of the anti-Tac MAb were constructed previously to form theanti-Tac dsFv molecule in which the two variable domains are heldtogether and stabilized by an interchain disulfide bond engineered atconserved framework residues (29, 30). To construct the scMHC-aTacVLmolecule the C-terminus of the scMHC molecule was connected to theN-terminus of anti-Tac VL using a 15-residues long flexible linker(Gly₄-Ser)₃ (SEQ ID NO:3). PCR amplified cDNAs of both molecules wereused in a two-step PCR overlap extension reaction in which the 3′-end ofscMHC was connected to the 5′-end of the VL gene. In the first step twothirds of the linker sequence and cloning sites were introduced toeither gene by using the oligonucleotides: scMHC-5:5′GGAAGCGTTGGCGCATATGATCC AGCGTACTCC-3′ (SEQ ID NO:11) and scMHC-3:5′-TCCTGAACCTCCGCCACCGGACCCTCCTCCGCCCTCCCATCTCAGGGT-3′ (SEQ ID NO:12),which introduce an NdeI restriction site at the 5′-end of the scMHC geneand two third of the linker at the 3′-end. The anti-Tac VL gene was PCRamplified with the oligonucleotides: VL-Tac-5:5′-TCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGCAAATTGTTCTCACC-3′ (SEQ ID NO:13)and VL-Tac-3: 5′-GCAGTAAGGAA TTCATTAGAGCTCCAGCTTGGT-3′ (SEQ ID NO:14) tointroduce two third of the linker at the 5′-end of the VL gene and anEcoRI cloning site at the 3′-end. In a second assembly step the two PCRproducts were combined in a 1:1 ratio (50 ng each) to form a PCR overpapextension reaction using the primers scMHC-5 and VL-Tac-3 for theassembly of scMHC-aTacVL construct. The PCR product was subsequentlysubcloned into the pET-based expression vector pULI7 (49) using the NdeIand EcoRI restriction sites. The anti-Tac VH gene for making theanti-Tac dsFv fragment was subcloned into pULI7 as previously described(29).

Expression, Refolding and Purification of B2M-aTac(dsFv)-PeptideComplexes:

The components of the B2M-aTac(dsFv); the scMHC-aTacVL and aTac VH, wereexpressed in separate BL21 (λDE3) cells (Novagen, Madison, Wis.). Uponinduction with IPTG, large amounts of insoluble recombinant proteinaccumulated in intracellular inclusion bodies. Inclusion bodies of eachcomponent were isolated and purified from the induced BL21 cells aspreviously described (29, 49). Briefly, cell disruption was performedwith 0.2 mg/ml of lysozyme followed by the addition of 2.5% TRITON X-100and 0.5 M NaCl. The inclusion bodies pellets were collected bycentrifugation (13,000 RPM, 60 minutes at 4° C.) and washed 3 times with50 mM Tris buffer, pH 7.4, containing 20 mM EDTA. Expression of eachrecombinant protein component in isolated and purified inclusion bodieswas determined by analyzing a sample on SDS-PAGE as shown in FIG. 2B.The isolated and purified inclusion bodies were solubilized in 6 MGuanidine HCl, pH 7.4, followed by reduction with 65 mM DTE. Solubilizedand reduced inclusion bodies of the scMHC-aTacVL and aTacVH, mixed in a1:2 molar ratio, were refolded by a 1:100 dilution into aredox-shuffling buffer system containing 0.1 M Tris, 0.5 M Arginine,0.09 mM Oxidized Glutathion, pH 10.0, in the presence of a 5-10 molarexcess of the HLA-A2-restricted peptides. The final proteinconcentration in the refolding was 50 μg/ml. After refolding the proteinwas dialyzed against 100 mM Urea, 20 mM Tris, pH 7.4, followed bypurification of soluble scMHC-aTac(dsFv)-peptide complexes byion-exchange chromatography on Q Sepharose column (7.5 mm innerdiameter×60 cm length, Pharmacia) applying a salt (NaCl) gradient (0-0.4M). Peak fractions containing scMHC-aTac(dsFv) were then subjected tosize-exclusion chromatography (TSK3000) for further purification andbuffer exchange to PBS.

ELISA:

Immunoplates (Falcon) were coated with 10 μg/ml purified p55 antigen(overnight at 4° C.). Plates were blocked with PBS containing 2% skimmilk and then incubated with various concentrations ofB2M-aTac(dsFv)-peptide (90 minutes at room temperature). Binding wasdetected using the anti-HLA conformational dependent antibody W6/32 (60minutes, room temperature, 10 μg/ml). The reaction was developed usinganti-mouse IgG-peroxidase. Rabbit anti-Tac antibody was used as apositive control, followed by anti-rabbit peroxidase.

Flow Cytometry:

Cells were incubated with B2M-aTac(dsFv)-peptide complexes (60 minutesat 4° C. in 300 μl, 25 μg/ml) washed and incubated with the anti-HLA-A2MAb BB7.2 (60 minutes at 4° C., μg/ml). Detection was with anti-mouseFITC. Human anti Tac (10 μg/ml) was used as positive control todetermine the expression of the p55 antigen followed by incubation withanti human FITC labeled antibody. Cells were subsequently washed andanalyzed by Beckman FACScaliber flow cytometer.

CTL Clones and Stimulation:

CTL clones specific for the melanoma gp100-derived peptides wereprovided by Drs. Steven Rosenberg and Mark Dudley, Surgery Branch,National Cancer Institute, NIH. These CTL clones were generated bycloning from bulk cultures of PBMCs from patients receiving peptideimmunizations (26). CTL clones were expanded by incubation withirradiated melanoma FM3D cells (as a source of antigen) and theEBV-transformed JY cells (B-lymphoblasts as antigen-presenting cells).The stimulation mixture contained also the OKT3 antibody (30 ng/ml) and50 IU/ml of IL-2 and IL-4.

Cytotoxicity Assays:

Target cells were cultured in 96 well plate (2−5×10³ cells per well) inRMPI+10 FCS. Cells were washed and incubated with methionine andserum-free medium for 4 hours followed by incubation (over night) with15 μCi/ml of ³⁵S-methionine (NEN). After 3 hours incubation withB2M-aTac(dsFv)-peptide complexes (at 37° C., 10-20 μg/ml), effector CTLcells were added at target:effctor ratio as indicated and incubated for8-12 hours at 37° C. Following incubation, ³⁵S-methionine release fromtarget cells was measured in a 50 μl sample of the culture supernatant.All assays were performed in triplicates. The percent specific lysis wascalculated as follows: [(experimental release−spontaneousrelease)/(maximum release−spontaneous release)]×100. Spontaneous releasewas measured as ³⁵S-methionine released from target cells in the absenceof effector cells, and maximum release was measured as ³⁵S-methioninereleased from target cells lysed by 0.1 M NaOH.

Experimental Results

Design of B2M-antiTac(dsFv):

Recently a construct encoding a soluble single-chain MHC (scMHC) wasgenerated in which the human β-2 microglobulin gene is linked to thethree extracellular domains (α1, α2 and α3) of the HLA-A2 heavy chaingene (aa 1-275) through a 15-amino acid-long flexible linker (24, 25 andWO 01/72768, which is incorporated herein by reference). These scMHCmolecules were expressed in E. coli as intracellular inclusion bodiesand upon in vitro refolding in the presence of HLA-A2-restricted tumorassociated or viral peptides they form correctly folded and functionalscMHC-peptide complexes and tetramers (24, 25, WO 01/72768). ThesescMHC-peptide complexes have been characterized in detail for theirbiochemical and biophysical characteristics as well as for theirbiological activity and found to be functional (24, 25, WO 01/72768).Most importantly, they were able to bind and stain tumor-specific CTLlines and clones. Shown in FIGS. 1A-H are the construction andreactivity of these scMHC-peptide complexes, in the form of scMHCtetramers, with CTLs specific for the melanoma differentiation antigengp100 epitopes G9-209M and G9-280V (26). These peptides are modified atthe MHC anchor positions 2 (in G9-209M) and 9 (in G9-280V) to improvethe binding affinity to HLA-A2 (27). The CD8⁺ CTL clones (FIGS. 1A and1D) R6C12 and R1E2 were stained intensively (80-95%) and specificallywith the G9-209M and G9-280V-containing scMHC tetramers, respectively(FIGS. 1B and 1E). As specificity control, the G9-209M-specific R6C12and G9-280V-specific R1E2 CTLs were not stained by G9-280V and G9-209MscHLA-A2 tetramers, respectively (FIGS. 1C and 1F). These CTLs alsoreacted with a similar intensity with the wild-type unmodified epitopesG9-209 and G9-280 (data not shown).

To generate the B2M-aTac(dsFv) molecule which targets the scMHC moleculeto cells through the use of an antibody Fv fragment, at the C-terminusof the HLA-A2 gene, was fused the light chain variable domain (VL) geneof the humanized anti CD25 (also known as Tac, p55, IL-2R α subunit)monoclonal antibody anti-Tac (28) (FIG. 2A). The heavy chain variabledomain (VH) is encoded by another plasmid to form a disulfide-stabilizedFv antibody fragment (dsFv) in which the VH and VL domains are heldtogether and stabilized by an interchain disulfide bond engineeredbetween structurally conserved framework residues of the Fv (FIGS. 2A,2E and 2F) (29,30). The positions at which the cysteine residues areplaced were identified by computer-based molecular modeling; as they arelocated in the framework of each VH and VL, this location can be used asa general method to stabilize all Fvs without the need for furtherstructural information. Many dsFvs have been constructed in the past fewyears, which have been characterized in detail and found to be extremelystable and with binding affinity as good as other forms of recombinantantibodies and in many cases even improved (30, 31).

Construction, Expression and Purification of B2M-antiTac(dsFv):

To generate the B2M-aTac(dsFv) molecule, two T7 promoter-basedexpression plasmids were constructed (see also Materials andExperimental Methods section hereinabove); the scMHC molecule fused toanti-Tac VL domain (B2M-aTacVL) is encoded by one plasmid and theanti-Tac VH domain is encoded by the second. In both plasmids the VL andVH domains contain a cysteine which was engineered instead of aconserved framework residue to form a dsFv fragment (30). The expressionplasmid for the B2M-aTacVL was generated by an overlap extension PCRreaction in which the HLA-A2 and VL genes were linked by a flexible15-amino acid—long linker of [(gly₄-ser)₃, (SEQ ID NO:3)] which isidentical to the linker used to connect the J32-microglobulin and HLA-A2genes in the scMHC construct (24, 25, WO 01/72768). The construction ofthe expression plasmid for the anti-Tac VH domain was describedpreviously (29). The two plasmids were expressed separately in E. coliBL21 cells. Upon induction with IPTG, large amounts of recombinantprotein accumulated in intracellular inclusion bodies. SDS-PAGE analysisof isolated and purified inclusion bodies demonstrated that recombinantproteins with the correct size constituted 80-90% of total inclusionbodies protein (FIG. 2B). The inclusion bodies of each component wereisolated separately, solubilized, reduced, and refolded in arenaturation buffer which contained redox-shuffling andaggregation-preventing additives, in the presence of HLA-A2-restrictedpeptides derived from the melanoma differentiation antigen gp100 T cellepitopes G9-209M and G9-280V (32-34, 27). The solubilized and reducedcomponents, B2M-aTacVL and anti-TacVH were mixed in a 1:2 molar ratio inthe presence of a 100-fold molar excess of the HLA-A2 restrictedpeptide. scMHC-peptide complexes and antibody Fv-fusion proteinsgenerated previously using this refolding protocol were found to befolded correctly and functional (24, 25, 30). B2M-aTac(dsFv)/peptidemolecules (complexes) were purified from the refolding solution byion-exchange chromatography using Q-Sepharose columns. As shown in FIG.2C, non-reducing SDS-PAGE analysis of peak fractions eluted from theMonoQ column revealed the presence of monomeric B2M-aTac(dsFv) moleculeswith the correct molecular weight of about 67 kDa. These factionscontained also B2M-aTacVL single-domain molecules that were not pairedwith the VH. These single-domain B2M molecules are difficult to separatefrom the B2M-dsFv molecules because, as also previously shown with otherdsFv-fusion proteins, VL-fusions folding is very efficient and theproduct is quite soluble. However, the contamination with thesingle-domain B2M molecules did not interfere with subsequent analysesof the soluble B2M-aTac(dsFv) molecule. To confirm the correct formationof the dsFv fragment, a reducing SDS-PAGE analysis was performed inwhich the B2M-dsFv molecule was separated to its components. Shown (FIG.2D) is the molecular form of the B2M-aTac(dsFv) after reductioncontaining the B2M-aTacVL and the VH domains. In any case, other sizeseparation techniques can be used to purify the B2M-aTac(dsFv) moleculeto homogeneity.

The ability of the B2M-aTac(dsFv) to bind its target antigen, the αsubunit of the IL-2 receptor (p55), was tested first by ELISA usingpurified p55. To monitor binding of the purified B2M-aTac(dsFv) top55-coated wells the monoclonal antibody w6/32 was used, whichrecognizes HLA molecules only when folded correctly and contain peptide.As shown in FIG. 2E, B2M-aTac(dsFv) binds in a dose dependent manner top55 which indicates that the two functional domains of the molecule, thescMHC effector domain and the antibody dsFv targeting domain, are foldedcorrectly, indicated by the ability of the dsFv moiety to bind thetarget antigen and the recognition of the scMHC by theconformational-specific anti-HLA antibody.

Binding of B2M-aTac(dsFv) to Target Cells:

To test the ability of the B2M-aTac(dsFv) molecule to coat and targetHLA-A2-peptide complexes on tumor cells, its binding to HLA-A2 negativetumor cells was tested by flow cytometry. First, A431 human epidermoidcarcinoma cells were used, that were stably transfected with the p55gene (ATAC4 cells) (35) and the staining of transfected versusnon-transfected parental cells was tested. The binding of B2M-aTac(dsFv)to the cells was monitored using an anti-HLA-A2 MAb BB7.2 andFITC-labeled secondary antibody. Expression of the p55 target antigenwas detected by the whole anti-Tac monoclonal antibody from which thedsFv fragment was derived. As shown in FIG. 3A, A431 cells do notexpress p55, however, the p55-transfected ATAC4 cells express highlevels of the antigen (FIG. 3B). Neither cell line was HLA-A2 positive(FIGS. 3C and 3D). When testing the binding of B2M-aTac(dsFv) to thesecells, FIGS. 3C and 3D show that ATAC4 cells gave a positive anti-HLA-A2staining only when preincubated with B2M-aTac(dsFv) (FIG. 3D), but A431cells were negative when preincubated with B2M-aTac(dsFv).

Next, the binding was tested of B2M-aTac(dsFv) to leukemic cells which,as shown in FIG. 3E, express the p55 antigen but lack HLA-A2 expression(FIG. 3F). As shown in FIG. 2F, the ATL leukemic HUT102W cellsexpressing p55, gave a positive anti-HLA-A2 staining when preincubatedwith the B2M-aTac(dsFv). Similar results were observed when leukemia(ATL) p55-positive, HLA-A2-negative CRII-2 cells were preincubated withthe B2M-aTac(dsFv) molecule (data not shown). These results demonstratethat B2M-aTac(dsFv) can bind to its antigen as displayed in the nativeform on the surface of cells. Most importantly, B2M-aTac(dsFv) could beused to coat HLA-A2 negative cells in a manner that was entirelydependent upon the specificity of the tumor targeting antibody fragmentrendering them HLA-A2 positive cells.

Induction of B2M-aTac(dsFv)-Mediated Susceptibility to CTL Lysis:

To test the ability of B2M-aTac(dsFv) to potentiate the susceptibilityof HLA-A2 negative cells to CTL-mediated killing radiolabeled targetcells were first incubated with B2M-aTac(dsFv) and then tested in a³⁵S-methionine-release assay in the presence of HLA-A2-restrictedmelanoma gp100-peptide-specific CTL. As shown in FIG. 4A, B2M-aTac(dsFv)induced an efficient CTL-mediated lysis of p55-positive HLA-A2 negativeATAC4 cells while the same B2M-aTac(dsFv) molecule did not have anyeffect and induced no lysis of A431 cells that do not express theantigen. A431 and ATAC4 cells alone did not exhibit any CTL-mediatedlysis (FIG. 4A). Incubation of ATAC4 cells with scMHC alone, not fusedto the dsFv targeting moiety, or with the anti-Tac antibody did notresult in any detectable potentiation of CTL-mediated lysis (data notshown). The capacity of G9-209M-peptide-specific CTLs to killB2M-aTac(dsFv)-preincubated ATAC4 cells (but not A431 cells) was asgood, and in many experiments better, as the efficiency of these CTLs tolyse melanoma FM3D cells which express high levels of HLA-A2 and thegp100 melanoma differentiation antigen (36) (FIG. 4B). To demonstratethe specificity of B2M-aTac(dsFv)-mediated CTL killing for theHLA-A2-restricted antigenic peptide used in the refolding of theB2M-aTac(dsFv) molecule, two CTL clones were used, specific for thegp100 major T cell epitopes G9-209M and G9-280V. As shown in FIG. 4C,p55-positive, HLA-A2-negative ATAC4 cells were lysed by theG9-209M-peptide-specific CTL clone R6C12 only when preincubated withB2M-aTac(dsFv) refolded with the G9-209M peptide but not with theG9-280V epitope derived from the same melanoma differentiation antigennor with B2M-aTac(dsFv) refolded around the HTLV-1 HLA-A2-restricted Tcell epitope TAX. Similarly, ATAC4 cells were killed by theG9-280V-specific CTL clone R1E2 only when preincubated withB2M-aTac(dsFv) refolded with the G9-280V epitope but not with theG9-209M or TAX peptides (FIG. 4D). Next, B2M-aTac(dsFv)-mediated CTLlysis of p55 expressing, HLA-A2 negative leukemic cells HUT102W andCRII-2 was tested. As shown in FIG. 4E, HUT102W and CRII-2 were notsusceptible to lysis by the HLA-A2-restricted CTL clones R6C12 and R1E2,specific for the G9-209M and G9-280V gp100 peptides, respectively.However, when these p55-positive, HLA-A2-negative target cells werepreincubated with the B2M-aTac(dsFv) molecule a significant potentiationfor CTL-mediated lysis was observed which was specific for the gp100peptide present in the B2M-aTac(dsFv) complex (FIG. 4E). B2M-aTac(dsFv)coated-HUT102W cells were efficiently killed by the G9-209M and G9-280Vpeptide-specific R6C12 and R1E2 CTL clones, respectively and CRII-2cells were lysed by the R1E2 CTL clone. Control non-melanoma HLA-A2positive and negative target cells that do not express p55 did notexhibit any detectable susceptibility to lysis by the melanoma-specificCTL clones weather coated or not with the B2M-aTac(dsFv) molecule (datanot shown). These results clearly demonstrate, in vitro, the conceptthat the B2M-aTac(dsFv) construct can be used efficiently forantibody-guided, tumor antigen-specific targeting of MHC-peptidecomplexes on tumor cells to render them susceptible to lysis by relevantCTLs and thus, potentiate anti-tumor immune responses.

In Vivo Activity of B2M-aTac(dsFv):

To initially evaluate the in vivo activity of B2M-aTac(dsFv) in a humantumor model, a win-type assay in which ATAC4 cells were mixed with R6C12CTLs specific for the G9-209M gp100-derived peptide was performed in thepresence or absence of the B2M-aTac(dsFv) molecule. The mixture wasinjected subcotaneously to nude mice and formation of human xenograftsin the animals was followed. As shown in FIG. 5, ATAC4 cells generatedxenografts in nude mice 10-12 days after subcutaneous injection.

A mixture of ATAC4 and R6C12 CTLs did not exhibit any significant effecton tumor growth. However, when IL-2 receptor expressing ATAC4 cells weremixed with B2M-aTac(dsFv) and R6C12 CTLs complete inhibition of tumorgrowth was observed indicating the efficient B2M-aTac(dsFv)-induced,CTL-mediated, killing of ATAC4 target cells in vivo. In vitro results(FIGS. 4A-E) confirmed that the amount of B2M-aTac(dsFv) and theeffector to target ratio used for the in vivo assay resulted in maximallysis of ATAC4 target cells (95-100% killing). Parental IL-2 receptornegative A431 cells mixed with R6C12 CTLs in the presence or absence ofB2M-aTac(dsFv) generated tumors efficiently, whereby no effect on tumorgrowth was observed (not shown).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of selectively killing a tumor cell in apatient, the cell presenting an antigen, the method comprisingadministering to the patient a molecule which comprises consecutiveamino acids present in the following consecutive segments, beginning atthe amino terminus of the molecule: (i) a human MHC-restricted peptidecapable of eliciting a memory response when present in the molecule,(ii) a first peptide linker, (iii) a human β-2 microglobulin, (iv) asecond peptide linker, (v) a human MHC class I molecule, and (vi) anantibody targeting domain which comprises an association of a heavychain variable region and a light chain variable region, wherein saidantibody is specific to said antigen, wherein the consecutive aminoacids which correspond to segments (v) and (vi) are linked to each otherdirectly by a peptide bond or by a third peptide linker and wherein thecarboxyl terminus of each of segments (i) through (v) is covalentlylinked to the amino terminus of segments (ii) through (vi),respectively, whereby said molecule initiates a CTL mediated immuneresponse against said cell, thereby selectively killing the tumor cell.2. The method of claim 1, wherein the human MHC-restricted peptide ofsegment (i) is from a viral protein.
 3. The method of claim 2, whereinthe viral protein is CMV.
 4. The method of claim 2, wherein said viralprotein is from an influenza.
 5. The method of claim 1, wherein segment(v) is linked either directly by a peptide bond or by a third peptidelinker to the light chain variable region of segment (vi).
 6. The methodof claim 1, wherein the heavy chain variable region and light chainvariable region are linked to each other by intermolecular disulfidebonds.
 7. The method of claim 1, wherein the heavy chain variable regionand light chain variable region are linked to each other by a fourthpeptide linker.
 8. The method of claim 1, wherein said antigen is EGFR.9. The method of claim 1, wherein said antigen comprises CD25.
 10. Themethod of claim 8, wherein said tumor cell is a lung cancer cell, abrain tumor cell or a breast cancer cell.